This invention relates to polyacrylamide gel electrophoresis and in particular, to a method of visualizing proteins in a polyacrylamide gel.
Polyacrylamide gel electrophoresis is a well-known technique for determining the molecular weight of a protein and for separating proteins on the basis of their molecular weight. Electrophoresis in the absence of any denaturing reagent (native-PAGE) results in separation on the basis of charge and size. It gives an estimation of the size of the folded protein by reference to proteins of known size. In order to determine the molecular weight of the polypeptide chain it is necessary to carry out the electrophoresis in the presence of the anionic detergent sodium dodecyl sulphate (SDS-PAGE). This detergent not only completely unfolds the protein but interacts with the unfolded chain such as to give a constant charge density. This means that separation is only based upon molecular weight. Calibration of the gel with marker proteins of known molecular weight allows estimation of the molecular weight of unknown proteins.
Visualization of the proteins separated by SDS-PAGE is typically carried out by staining the gel with Coomassie brilliant blue or Amido black dyes. Other non-specific visualization techniques include silver precipitation or staining with fluorescent compounds. Coomassie blue staining is the most common technique and, similar to other prior art techniques, typically involves several hours of protein fixation, staining and destaining. There have been many attempts to provide shorter staining protocols.
The use of fluorescence for protein detection is of course well established in biochemistry. Preelectrophoretic labeling of proteins with UV-excitable fluorophores, such as FITC (flourescein isothiocyanate) or bromobimane compounds (1) followed by postelectrophoretic visualization under UV light has been successfully used for many years. Also, several methods have been developed for postelectrophoretic fluorescent labeling with stains such as 1-aniline-8-naphthalene sulfonate (2) and 0-phthaladehyde (3). All these methods involve lengthy labeling steps and each of them has intrinsic limitations such as altered electrophoretic mobility on native PAGE in the case of preelectrophoretic labeling or low sensitivity in the case of postelectrophoretic staining (4). Recently two new fluorescent dyes, SYPRO red and SYPRO orange, have been introduced to detect proteins in SDSPAGE (5). Although quite sensitive, their use is expensive, somewhat time-consuming, and dependent on the presence of SDS.
There is still a need in the art for further improvements in fluorescent protein visualization.